(A) Characterization of the human inducible nitric oxide synthase promoter: Nitric oxide production contributes to the initiation and modulation of the inflammatory response to lipopolysaccharide (LPS) and cytokines. Cytomix (CM) (10ng/ml TNF-alpha, 100 U/ml IFN-gamma, and 0.5 ng/ml IL-1beta) activated an 8.3-kb human inducible nitric oxide synthase (hiNOS) promoter via two NFkappaB and two AP-1 sites. To define the signaling pathways involved in the regulation of the hiNOS gene by LPS and CM, the 8.3-kb hiNOS promoter, linked to a luciferase reporter, was transfected into A549 cells. Thirty-six hours after transfection, cells were incubated with inhibitors of signal transduction pathways for 1 h and then stimulated for 6 h in the presence of inhibitor with 10 mug/ml LPS plus 100 U/ml IFN-gamma (LPS/IFN-gamma) or CM. CM or LPS/IFN-gamma, but not LPS (0.1-10 mug/ml) or IFN-gamma (100/U/ml) alone, stimulated the hiNOS promoter. In the presence of CM or LPS/IFN-gamma, the p38 MAP kinase inhibitor SB203580 (5-40 muM) and the MEK1 inhibitor PD98059 (5-50 muM) each caused 50% attenuation of hiNOS promoter activity. LPS/IFN-gamma- as well as CM-stimulated promoter activity was eliminated when both inhibitors were present. The phosphatidylcholine phospholipase C inhibitor D609 (10-100 muM, presumably by preventing protein kinase C activation by diacylglycerol), the p42 MAP kinase inhibitor AG126(10-150 muM), and the JAK2 inhibitor AG490 (5-100 muM, presumably by interfering with IFN-dependent signaling) abolished both CM- and LPS/IFN-gamma-induced promoter activity. These data are consistent with the conclusions that (1) LPS and IFN-gamma act synergistically to activate hiNOS; (2) induction of promoter activity by CM and LPS/IFN-gamma is dependent on activation of ERK, and p42 and p38 MAP kinases; and (3) release of diacylglycerol is an early signaling event that leads to hiNOS promoter activation. (B) Regulatory properties of human inducible nitric oxide synthase: Human inducible nitric oxide synthase (iNOS) is active as a homodimer. The region encoded by exons 8 and 9, containing 94 amino acids, is critical for iNOS dimer formation and nitric oxide (NO) production. In this study, alanine scanning mutagenesis was used to identify the amino acids in that domain critical for iNOS activity. Based on the crystal structure of the cognate domain of mouse iNOS, 16 conserved amino acids, judged to be involved in catalytic activity and/or dimer formation, were analyzed. Using full length human iNOS cDNA as a template, site-directed mutagenesis was employed to construct 16 iNOS mutants each with an alanine replacing one of the 16 selected amino acids. The resulting iNOS mutants were characterized following their expression in 293 human epithelial cell line. All iNOS mutants yielded iNOS protein as detected by Western analysis using specific iNOS antibody. Four iNOS mutants with alanine substituted for Trp260, Asn261, Tyr267, or Asp280, respectively, did not generate NO. Dimer formation was tested by SDS-PAGE at 4 degrees C, followed by immunoblotting. As expected, wild type iNOS and all 12 NO-producing iNOS mutants migrated both as monomers and dimers. However, NOS mutants with alanine replacing Trp260, Asn261, or Tyr267 migrated only as monomers suggesting that their inability to produce NO is related to a defect in dimer formation. Interestingly, the Asp280 mutant retained the ability to dimerize, indicating that it represents an inactive form of an iNOS dimer. These data identify four amino acids critical for iNOS activity and/or dimer formation.